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Interactions of whey protein isolate and human saliva, as related to the astringency of whey protein beverages : a thesis in partial fulfilment of the requirement of the degree of Master of Technology in Food Technology at Riddet Institute, Massey University, New Zealand

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Abstract

Interactions between 3 different proteins (lactoferrin, beta-lactoglobulin and Whey
Protein Isolate) and human saliva were determined. Lactoferrin and whey proteins
are known to be astringent at low pH. Astringency is defined as the tactile sensation,
mainly on the tongue, caused by astringent compounds when in contact with human
saliva. Proline-rich proteins are already known to be directly involved in the
astringency of polyphenols. Whey proteins do not contain polyphenols. However,
because whey proteins at low pH develop an astringent sensation when consumed,
it was expected to detect proline-rich proteins in the interaction between Whey
Protein Isolate (WPI) and saliva as well.
The protein solutions were adjusted to different pH-levels, ranging from neutral to
high acidic, where a part of each protein solution was heat-treated. All solutions were
mixed with human saliva in the same ratio (w/w). One part of all mixtures was
pH-readjusted. Additionally, WPI model solutions were prepared, adjusted to
different pH-levels, heat-treated and then consumed by voluntary participants, who
swirled each solution in their mouth for at least 10 seconds. These mixtures of WPI
and saliva were collected for further analysis. After consuming the WPI model
solutions, followed by rinsing the mouth with water, tongue swabs were taken to
determine the particle sizes and zeta-potentials of the remaining material on the tongue.
Control tongue swabs of the clean tongue were taken by the participants before any
consumption of the WPI model solutions.
All mixtures as well as lactoferrin, beta-lactoglobulin (beta-lg), WPI and saliva on their own,
were analysed for particle size, zeta-potential and turbidity, which may give an indication
for possible aggregation/precipitation of the proteins as well as the analysis of the
SDS-PAGE profile of the sediments of the sample mixtures. Saliva is negatively charged between neutral pH and 3.0, whereas lactoferrin has a
positive charge below pH 8.0. WPI has a positive charge below pH 5.1; the same
applies to beta-lg. None of the proteins themselves showed aggregation/precipitation at pH-levels 6.8, 3.6, 3.4, 3.0, 2.5 or 2.0. However, after the proteins were mixed with
saliva, the pH of mixtures shifted towards neutral pH.
The mixtures of lactoferrin (unheated/heat-treated) and saliva neither showed any
significant increases in particle size nor the presence of turbidity. Salivary proteins
were not detected in any mixtures at any observed pH either, despite the known fact
that lactoferrin causes astringency. The mixtures of beta-lg (unheated/heated) and
saliva displayed high particle sizes below final pH 3.6, whereas the high turbidities of
both mixtures were measured between final pH 3.6 and 3.4. Furthermore, only at
final pH 2.8 were salivary proteins (mainly glycosylated proline-rich proteins and
alpha-amylase) detected. However, higher concentrations of salivary proteins were
measured when heat-treated beta-lg was mixed with saliva. The mixtures of WPI and
saliva presented the strongest interaction compared to lactoferrin and beta-lg. High
aggregation/precipitation occurred in the mixtures between pH 4.3 and 3.0, where
significantly high particle sizes and turbidities were detected.
The pH-readjusted mixtures of lactoferrin/beta-lactoglobulin/WPI and saliva showed
similar values in particle size and turbidity as the mixtures of the proteins and saliva
without pH-readjustment at similar pH-values. Furthermore, the pH-readjusted
mixtures of the proteins and saliva showed in their sediments the presence of
alpha-amylase and glycosylated proline-rich proteins.The mixtures of heat-treated WPI and saliva, collected from the mouth after taking a
sip (ratio unknown), revealed that the strongest interactions occurred when
WPI-solutions were adjusted to pH 3.6 and 3.4. Similar observations were made for
heat-treated WPI-solutions, which were adjusted to pH 3.6 and 3.4, when mixed with
saliva 1:1 (w/w). However, additionally to the glycosylated proline-rich proteins and
alpha-amylase, faint bands of mucin as well as basic proline-rich proteins were detected
in the mixtures collected from the mouth.
The proteins of the material remaining on the tongue followed the consumption of
WPI-solutions and rinsing with water showed that the particle size measurementswere not reliable. However, pH-levels between 6.8 and 5.7 occurred and negative
charges were measured on the tongue after rinsing the mouth twice with water.
The strongest interactions between the proteins and human saliva occurred when
the proteins, in particular beta-lg and WPI, were positively charged and then mixed with
saliva (negative charge). Concluding from that it is suggested that electrostatic
interactions may cause the astringent sensations. However, since no evidence could
be found that salivary proteins were involved in the interaction between lactoferrin
and saliva (without pH-readjustment), it is suggested that other interactions than
electrostatic interactions cause the astringent sensation of lactoferrin.